Production of 5&#39;-nucleotides



United States Patent 3,223,592 PRODUCTION OF -NUCLEOTIDES KinichiroSakaguchi, Tokyo, and Akira Kuninaka,

Choshishi, Chihaken, Japan, assignors to Yamasa Shoyu Kabushiki Kaisha(doing business as Yamasa Shoyu Co., Ltd.), Choshi, Japan, a Japanesecorporation No Drawing. Filed Aug. 22, 1958, Ser. No. 756,541

Claims priority, application Japan, Sept. 27, 1957, Bil/23,698; Apr. 28,1958, 33/11,586 2 Claims. (Cl. 195-28) This invention relates to aprocess for producing the solution containing 5'-nucleotides(adenosine-5'-monophosphate, guanosine-5-monophosphate,uridine-5'-monophosphate, cytidine-5-monophosphate,inosine-5'-monophosphate, xanthosine-5'monophosphate) from ribonucleicacid by microbial 5-phosphodiesterase action, and to an application ofthe 5'-nucleotides as special seasonings. The object of this inventionis to produce flavorous 5-nucleotides, which were so far preparedgenerally only by organic synthesis or by extraction from tissues ofvarious organisms such as mammalian muscle, eco nomically and in goodyield from ribonucleic acid, using the enzymes of microorganisms.

Chemical degradation of ribonucleic acid results in formation of 3- and2-nucleotides and does not result in formation of 5-nucleotides.Furthermore general ribonucleodepolymerases, without distinction of thekind of origins, degrade ribonucleic acid into 3' (or 2')-nucleotidesbut not into 5'-nucleotides. Only so-called unspecificphosphodiesterases from snake venom or intestinal mucosa degraderibonucleic acid into 5-nucleotides. However, it is very difiicult toobtain a large amount of these enzymes. 5-nucleotides can be produced bymeans or organic synthesis but said process is very troublesome and noteconomical too. Thus, hitherto, the production of 5'-nucleotides wasvery difficult, and especially economical mass production thereof wasquite impossible.

We have found that some strains of bacteria, yeasts, and molds contain5-phosphodiesterases which specifically hydrolyze the 5-phosphodiesterlinkages in ribonucleic acid and produce four 5'-nucleotides:adenosine-S-monophosphate, guanosine-S'-monophosphate,cytidine-S'-monophosphate, and uridine-5'-monophosphate. Especially theseveral strains which belong to the following genuses have beenrecognized to contain strong 5'-phosph0diesterase: Bacillus,Streptomyces, Torula, Zygosaccharomyces, Penicillium, and Aspergillus.Thus the basis of the production of 5'-nucleotides by microorganismsaccording to the present invention has been established for the firsttime.

This invention has been accomplished on the basis of the aboveconfirmation. Therefore, the present invention provides a process forthe production of 5-nucleotides which is characterized in this thatribonucleic acid is degraded into 5'-nucleotides by 5'-phosphodiesterasewhich is contained in living cells, dry cells, culture filtrates or cellextracts of microorganisms described above. The microorganismscontaining 5-phosphodiesterase are able to be grown on either solidmedia or liquid media. For economical mass production, however, liquidmedia are more appropriate. As the components of the culture medium, theconventional carbon and nitrogen sources and several inorganic salts maybe employed effectively. This invention includes both one step methodand two step method. In one step method, both growing of microorganismand enzymic degradation of ribonucleic acid are carried outsimultaneously, em-

Patented Dec. 14, 1965 ploying culture medium containing ribonucleicacid. In two step method, growing of microorganism and enzymicdegradation of ribonucleic acid are carried out separately.

According to the present invention, it is not necessary to purifyribonucleic acid before its enzymic degradation. Crude solutioncontaining ribonucleic acid, such as yeast extracts, may be used as anappropriate starting material. Furthermore microbial cells cultivatedfor producing 5- phosphodiesterase are effectively utilized too as asource of ribonucleic acid.

Free 5'-nucleotides or their alkali salts obtained by the processing asdescribed above enhance or increase the flavor of the foods, beverages,and seasonings in which they are placed. This flavoring action is causedby the synergy between 5-nucleotides and amino acids or organic acids.According to our discovery purine and pyrimidine bases, theirnucleosides, and their 2'- and 3-nucleotides have little flavor, while5'-nucleotides, especially inosine-S'-monophosphate,guanosine-5-monophosphate, and xanthosine-S'-monophosphate, have veryagreeable good taste. Furthermore, there is specific synergy in tastebetween 5-nucleotides and amino acids or organic acids. Among variousamino acids, glutamic and aspartic acids were recognized to beespecially effecctive in the synergy with 5-nucleotides. General foods,beverages, and seasonings contain considerable quantity of amino acidsor organic acids as main flavoring components, but scarcely contain5-nucleotides. Therefore, it seems that the role of S'-nucleotides inflavoring is very important. For example the good taste of soups or meatextracts, containing small quantity of 5'-nucleotides, may be perhapscaused mainly by the synergy between 5'-nucleotides and amino acids.

This invention relates also to the application of 5'- nucleotides basedon the utilization of aforesaid synergy between 5-nucleotides and aminoacids or organic acids. The application of 5 -nucleotides according tothe present invention comprises adding one or more of 5-nucleotides togeneral foods or beverages such as meat products, soups, roux, vinegar,various dressings, sauces, curry powder and various drinks includingwine, to counteract the displeasing pungency which spoils the tastequalities of foods or beverages, and to enhance or increase the flavorspecifically according to the synergy between 5-nucleotides and aminoacids or organic acids present in the foods or beverages. In case ofapplication for the foods or beverages containing no amino acids, it ismore effective to add monosodium glutamic acid and 5'-nucleotidestogether. 5-nucleotides may be also employed to enrich specifically theseasonings containing amino acids. The bitter substances in the crudepreparations of 5-nucleotides can be readily removed by cation exchangeresin. Both crude and purified preparations of 5-nucleotides are useful.

According to the present invention, alkali salts of 5-nucleotides may bealso employed similarly as free 5'-nucleotides since there is nosignificant difference between their flavoring action.

The invention is illustrated but not limited by the following examples.

Example 1 50 ml. of an aqueous culture medium containing 5% of glucose,0.5% of polypeptone, 0.05% of monobasic potassium phosphate, 0.05% ofdibasic potassium phos phate, 0.04% of magnesium sulfate, and 0.04% ofcalcium chloride were sterilized and inoculated with a pure culture ofPenicillium citrinum. After surface culture at 30 C. for five days themycelial deck was separated from the culture broth, and washed withsterilized water. The washed mycelial deck was incubated with 50 ml. of

0.5% yeast ribonucleic acid solution containing 0.01 N sodium fluorideat 30 C. After 22.5 hours the deck was removed. The resulting reactionmixture was recognized to contain 70-80 mg. of mononucleotides, 80-90mg. of nucleosides, and 70-80 mg. of undepolymerized polynucleotides.The mononucleotides, which are contained in above mixture, wereidentified as cytidine-- monophosphate, adenosine-S'-monophosphate,inosine-5- monop'hosphate, uridine-S'-monophosphate, andguanosine-5'-monophosphate. The identification was carried out asfollows: 23 ml. of the reaction mixture were adjusted to pH 8.5 withstrong sodium hydroxide solution. 2.5 ml. of 20% barium acetate solutionwere added thereto. The precipitate of barium phosphate formed wasremoved. The supernatant was adjusted to pH 5.0 with .a small quantityof acetic acid. 1 ml. of mercuric acetate solution (20% in 2% aceticacid) was added. The precipitate was centrifuged, washed and suspendedin Water. Into the suspension hydrogen sulfide gas was introduced toseparate nucleotides. The mixture was filtered and the precipitate waswashed with hot water. The solution resulting from the washing wascombined with the supernatant and a portion of the combined solutionswas adjusted to pH 8.5, and charged into anion exchange resinDoweX-l-Cl-X-4 (200-400 mesh) column with a diameter of 1.0 cm. and 23cm. in height, and was eluted with 0.003 N (No. l-No. 216 test tubes)and 0.010 N hydrogen chloride (No. 2l7-No. 302 test tubes). Each 80drops of the eluate were collected into a test tube and optical densityat 260 mg of each eluate was read. Five ultraviolet absorbing fractionsA, B, C, D and E were obtained. The properties of these fractions aretabulated as follows.

medium was shaken on a reciprocating shaker at C. After 7 days culturefiltrates were concentrated in vacuo, and then dialyzed against runningwater over night. To the dialyzed solution 4 volumes of ethanol wereadded. The resulting precipitate, which was rich in 5'phosphodiesteraseactivity, was dried up in a desiccator and was employed as an enzymepreparation. From 1 litre of culture filtrates about 1 g. of thepreparation was obtained. 1 g. of this preparation was incubated with200 ml. of 5% ribonucleic acid at C. and pH 5.0. Under these conditionsphosphomonoeste-rase and adenyl deaminase were almost inactive, while5'phosphodiestera-se was recognized to be very active.

The reaction proceeds as described below.

Incubation time (min) 0 I 10 30 i 60 5'nucleotides formation fromribonucleic acid (percent) 27 68 96 97 Inorg. P. formation from5'nucleotides (percent) 3.0 3. 5 4. 5 5. 7

Example 3 100-500 mg. of the crude mixture of adeno=sine-5'- Nucleotidetraction obtained Standard substance Fraction A B C D E Cytidine-Adenosine- Adenosine- Inosine- Urldine- Guanosine- Mixture 3-mono-3-mono- 5'mono- 5'mono- 3-mono- 3-monooi 3- nuphosphate phosphatephosphate phosphate phosphate phosphate cleotldcs No of test tube 21-2443-54 121-122 221-237 264-281 Rm (Inn) 1 275 258 249 262 257 278 257 257250 262 257 258 Percent color development. pentoseorcinoi reaction:

7 min 78. 7 82.2 81. 6 80. 4 39.6 97. 2 95. 7 72. 8 100. 0 100. 0 94. 796.3 95.4 99. 0 92. 8 92. 1 100. 0 arbazole reaction. blue purple.blueblue purple. Distance 3 migrated from origin to anode side byelectrophoresis (0111.) 4. 9 4. 9 12. 5 14. 7 7. 7 4. 9 5. 0 4. 5 12.515.0 8. 8 14.8,8.7,4.7 NalOi-rosaniline reaction I Ultravioletabsorption spectra 01 standard substances were measured in 0.1 N H01.

2 The technique employed was essentially the same as that described Theresults shown in the table indicate that the ultraviolet absorbingsubstances which are contained in fractions A, B, C, D and E arecytidine-S-monophosphate, adenOsine-S -monophosphate, inosine-S'-monopho-sphate, uridine-S-monophosphate, andguanosine-5'-mor1ophosp'hate, respectively. It seems very probable thatinosine- 5'monophosphate recognized in this example was producedsecondarily from adenosine-S-monophosphate by the action of Henicilliumdeaminase. In this example the formation of xarithosine-S-monophosphatewas not recognized. However, this compound was easily obtainedenzymatically or chemically from guanosinc-5'-monophophate.

Example 2 For the formation of the strong 5'phosphodiesterase, shakingculture is more eifcctive than surface culture at least in case of thestrain employed in Example 1.

The culture medium employed in Example 1 Was inoculated with Penicilliumcitrinum. The inoculated growth by Albaum and Umbreit (J. Biol. Chem.,167, 369, (1047)). 3 Starting line was at 5 cm. from the end on thecathode side, and 26 cm. irom the end on the anode side.

Example 4 Monosodium glutamate was coated with purifiedinosine-5-monophosphate disodium salt or guanosine-5- monophosphoricacid. The ratio of monosodium glutamate to inosine-5-monophosphate orgua-nosine-5-monophosphate was 5-5:l. The resultant superior seasoningwas recognized to have remarkable flavoring properties for all kind ofdishes.

Example 5 To 120 g. of soup potage powder (corresponding to 1800 ml. offinal volume) 1 to 2 g. of purified inosine- 5-monophosphate disodiumsalt or guanosine-5'-monophosphoric acid were added. From the result-antenriched powder, remarkable fiavorous soup was prepared. Instead of thepurified nucleotide preparations each crude preparation or mixture of5'-nucleotides was also employed satisfactorily.

In case of soup consum-m being prepared inosine-5'- monophosphate,guanosine-S-monophosphate, and mixture of 5'-nuoleotides were employedeffectively too.

What we claim is:

1. A process for preparing 5'-nucleotides which compnises intimatelycontacting ribonucleic acid in an aqueous medium with a5-phosphodiesterase=containing enzyme produced by Penicillium citrinum,and recovering the 5'- nucleotides from the resulting medium.

2. A process for preparing 5-nucleotides which comprises growingPenicillium citrinum in an aqueous nutrient medium, recovering the5'-phosphodiesterase from the resulting fermentation broth, incubatingthe recovered 5'-phosphodiesterase in an aqueous medium containingribonucleic acid, and recovering the 5'-nucleotides from the resultingmedium.

References Cited by the Examiner UNITED STATES PATENTS OTHER REFERENCESCohn et al.: Journal of Biological Chemistry, vol. 203, July-August1953, pages 319 to 331.

Dixon et al.: Enzymes, published by Academic Press Inc., New York, 1958,pages 190, 191 and 690.

A. LOUIS MONACELL, Primary Examiner.

ABRAHAM WINKELSTEIN, TOBIAS E. LEVOW,

Examiners.

1. A PROCESS FOR PREPARING 5''-NUCLEOTIDES WHICH COMPRISES INTIMATELYCONTACTING RIBONUCLEIC ACID IN AN AQUEOUS MEDIUM WITH A5''-PHOSPHODIESTERASE-CONTAINING ENZYME PRODUCED BY PENICILLIUMCITRINUM, AND RECOVERING THE 5''NUCLEOTIDES FROM THE RESULTING MEDIUM.